Recombinant BCG tuberculosis vaccine designed to elicit immune responses to Mycobacterium tuberculosis in all physiological stages of infection and disease

ABSTRACT

A vaccine against  Mycobacteria tuberculosis  (Mtb) is provided. The vaccine comprises a recombinant Bacille Calmette-Guerin (BCG) subunit-based vaccine in which one or more Mtb antigens and one or more Mtb resuscitation or reactivation antigens are overexpressed, and in which at least a portion of the DosR regulon is up-regulated. The vaccine is protective against active Mtb infection both pre- and post-exposure to Mtb, and thus prevents disease symptoms due to the recurrence of a latent Mtb infection.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention generally relates to improved Mycobacterium tuberculosis (Mtb) vaccines that are successful in preventing the development of symptoms of tuberculosis, both pre- and post-exposure to Mtb. In particular, the invention provides an improved recombinant Bacille Calmette-Guerin (BCG) subunit-based vaccine in which one or more Mtb antigens and one or more Mtb resuscitation or reactivation antigens are overexpressed, and in which at least a portion of the DosR regulon is up-regulated.

2. Background of the Invention

The current prophylactic (pre-exposure) Mycobacterium tuberculosis (Mtb) vaccine Mycobacterium bovis (M. bovis) BCG, introduced over 60 years ago, efficiently protects against severe disease manifestation in children but fails to prevent the establishment of latent TB or disease reactivation of infection in adolescents and adults. Moreover, essentially all novel Mtb vaccines currently in clinical trials are designed as prophylactic rather than both prophylactic and therapeutic (post exposure) vaccines.

It is believed that Mtb progresses through a series of stages during its infectious cycle in man as a reaction to human immune responses and that each stage is orchestrated by a distinct genetic program which directs the expression of stage-specific antigens. If this concept is valid, then a truly comprehensive tuberculosis vaccine should include antigens representing each stage as well as antigens that are stage-independent. Latent tuberculosis (LTBI/latency) appears to be one such stage and current evidence suggests that Mtb adopts a unique physiological phenotype during latency characterized by bacteriostasis (non-replicating persistence), a switch from aerobic to anaerobic respiration, expression of the α-crystalline small chaperone protein (Acr/HspX) and increased resistance to several mycobacterial antibiotics.

Maintenance of the non-replicating persistence state, believed to be typical of Mtb in latent lesions, appears to depend on the continuous production of Th1 cytokines (IFNγ, IL-12 and TNFα) and nitric oxide and the localization of MTB within stable granulomas. However, the reactivation of latent Mtb infection, characterized by resumption of bacterial replication, inflammation and cavitation, can be promptly precipitated by immunosuppressive regimens (e.g., corticosterioids or TNFα-antagonist) and occurs in 5-10% of latently infected individuals, perhaps due to acquired tolerance to environmental mycobacteria, age, and, more significantly, HIV disease. This common clinical scenario and the proven role of the cellular immune system for the maintenance of latency lead to the conclusion that non-replicating persistence is a metastable phenotype determined by three interacting processes: bacterial replication within latent lesions is constrained by effectors of the cellular immune system; bacteria within latent lesions monitor the production of immune effectors; and, decreased production of immune effectors results in resumption of replication.

To date, no successful vaccines have been developed which confer immunity to infection by Mtb and at the same time treat or prevent the development of symptoms of TB after exposure to Mtb, or as a result of reactivation of latent infection. A recombinant BCG vaccine, engineered to elicit an immune response of this kind, might reduce reactivation rates in persons with subtle degrees of immunosuppression produced, for example, by senescence, diabetes, HIV disease, acquired tolerance to environmental mycobacteria or malnutrition. There is thus an ongoing need to develop new TB vaccines, and it would be particularly useful to develop a vaccine that can be used both prophylactically and for post-exposure treatment.

SUMMARY OF THE INVENTION

The present invention is based on the development of a novel recombinant Bacille Calmette-Guerin (rBCG) for use as a vaccine. The vaccine may be used prophylactically to prevent Mtb infection in naive individuals. However, the vaccine is also effective for treating individuals who have already been exposed to and/or infected by Mtb. The vaccine prevents the establishment of infection and likewise prevents the reactivation of latent Mtb in individuals that have been previously infected. The rBCG that is used in the vaccine preparations is genetically engineered to express “classical” Mtb antigens and antigens that are relevant to several stages of the Mtb life cycle, e.g. latency, reactivation and resuscitation. Thus, the immune response that is generated as a result of immunization with the vaccine protects the vaccinated individual from developing an active Mtb infection at any and all stages of exposure to Mtb. In particular, the rBCG overexpresses 1) one or more genes encoding Mycobacterium tuberculosis (Mtb) antigens that are known to elicit potent, protective immune responses to Mtb; and 2) one or more genes encoding at least one Mtb resuscitation or reactivation antigen. The antigen encoding sequences are located on an extrachromosomal element or are integrated into the chromosome of the recombinant BCG. In addition, expression of all or part of the Dos R regulon is up-regulated in the novel rBCG. The embodiment of the invention in which the antigen encoding sequences are integrated into the chromosome is depicted schematically in FIG. 1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic representation of the rBCG of the invention.

FIG. 2. Diagram of integration plasmid and expression cassette.

FIG. 3A-D. Immunoblots demonstrating the expression of chromosomally integrated antigen cassettes. A, Expression or Rv0867c from AERAS-407 precursor AFV-102pRC108, Lane 1=AFV-102pRC108, Lane 2=AFV-102; B, Overexpression of Ag85A and Ag85B from AERAS-407 precursor AFV-102RC108, Lane 1=AFV-102, Lane 2=AFV-102pRC108; C, Expression of Rv3407 from AERAS-407 precursor AFV-102pRC108, Lane 1=AFV-102, Lane 2=AFV-102pRC108; D, Expression of DosR (Rv3133c) from AERAS-407 precursor AFV-102pRC108, Lane 1=AFV-102, Lane 2=AFV-102pRC108.

FIG. 4. Diagram of a second integration plasmid and expression cassette.

FIG. 5A and B. Rv3804c amino acid sequence (A, SEQ ID NO: 1) and nucleotide sequence (B, SEQ ID NO: 2).

FIG. 6A and B. Rv1886c amino acid sequence (A, SEQ ID NO: 3) and nucleotide sequence (B, SEQ ID NO: 4).

FIG. 7A and B. Rv0867c amino acid sequence (A, SEQ ID NO: 5) and nucleotide sequence (B, SEQ ID NO: 6).

FIG. 8A and B. Rv1009c amino acid sequence (A, SEQ ID NO: 7) and nucleotide sequence (B, SEQ ID NO: 8).

FIG. 9A and B. Rv1884c amino acid sequence (A, SEQ ID NO: 9) and nucleotide sequence (B, SEQ ID NO: 10).

FIG. 10A and B. Rv2389c amino acid sequence (A, SEQ ID NO: 11) and nucleotide sequence (B, SEQ ID NO: 12).

FIG. 11A and B. Rv2450c amino acid sequence (A, SEQ ID NO: 13) and nucleotide sequence (B, SEQ ID NO: 14).

FIG. 12A and B. Rv2623c amino acid sequence (A, SEQ ID NO: 15) and nucleotide sequence (B, SEQ ID NO: 16).

FIG. 13A and B. Rv0288c amino acid sequence (A, SEQ ID NO: 17) and nucleotide sequence (B, SEQ ID NO: 18).

FIG. 14A and B. Rv2626c amino acid sequence (A, SEQ ID NO: 19) and nucleotide sequence (B, SEQ ID NO: 20).

FIG. 15A and B. Rv2005c amino acid sequence (A, SEQ ID NO: 21) and nucleotide sequence (B, SEQ ID NO: 22).

FIG. 16A and B. Rv1996c amino acid sequence (A, SEQ ID NO: 23) and nucleotide sequence (B, SEQ ID NO: 24).

FIG. 17A and B. Rv0685c amino acid sequence (A, SEQ ID NO: 25) and nucleotide sequence (B, SEQ ID NO: 26).

FIG. 18A and B. Rv0824c amino acid sequence (A, SEQ ID NO: 27) and nucleotide sequence (B, SEQ ID NO: 28).

FIG. 19A and B. Rv2029c amino acid sequence (A, SEQ ID NO: 29) and nucleotide sequence (B, SEQ ID NO: 30).

FIG. 20A and B. Rv2627c amino acid sequence (A, SEQ ID NO: 31) and nucleotide sequence (B, SEQ ID NO: 32).

FIG. 21A and B. Rv2744c amino acid sequence (A, SEQ ID NO: 33) and nucleotide sequence (B, SEQ ID NO: 34).

FIG. 22A and B. Rv3347c amino acid sequence (A, SEQ ID NO: 35) and nucleotide sequence (B, SEQ ID NO: 36).

FIG. 23A and B. Rv1130c amino acid sequence (A, SEQ ID NO: 37) and nucleotide sequence (B, SEQ ID NO: 38).

FIG. 24A and B. Rv1169c amino acid sequence (A, SEQ ID NO: 39) and nucleotide sequence (B, SEQ ID NO: 40).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

The present invention provides a novel rBCG for use in Mtb vaccine preparations. The rBCG is genetically engineered to overexpress 1) one or more Mtb antigens, which may include so-called “classical” Mtb antigens such as Rv1886c-Ag85B (“85B”) and Rv3804c-Ag85A (“85A”), among others; and 2) at least one Mtb resuscitation/reactivation antigen. In addition, in the rBCG of the invention, the DosR regulon, or a portion thereof, is up-regulated. The DosR regulon, comprised of 48 genes, is typically activated in Mtb as a result of a post-infection drop in O₂ tension (hypoxia).

The Mtb antigens that are selected for inclusion in the rBCG of the invention are generally those which are known or predicted to elicit a protective immune response in individuals exposed to the antigens. Various criteria may be used to select suitable antigens, including but not limited to: the observation that individuals with immune responses to the antigen are able to control Mtb infection, determination that the antigen contains one or more T-cell epitopes via an immunoinformatics analysis (e.g. using a program such as that which is found at the website of the Technical University of Denmark: cbs.dtu.dk/services/NetCTL, in which the analysis is based on the identification of proteosome cleavage sites, endoplasmic reticulum (ER) transport efficiency, major histocompatibility (MHC) class 1 binding affinity, etc.); an analysis of experimental evidence based on an in-depth literature search; sorting or ranking of results according to various parameters such as macrophage survival/persistence; up-regulation of expression by the two-component system MprAB; response to hypoxia; involvement in dormancy; expression in lung tissue; genes with promoters that are co-regulated with Rv2031 (Acr); proteins that are secreted (which are more accessible to the immune system); presence of repeats (many virulence associated proteins have amino acid repeat domains); ability to act as a B-cell immunogen; cell wall associated or cell walls biogenesis (membrane-exposed/associated proteins are considered to be more accessible to the immune system; existing vaccine efficacy data; uniqueness to Mtb; etc.) Thus, antigens may be selected based on experimental data demonstrating their efficacy, or alternatively (or in addition) such antigens may be selected based on their predicted abilities. Those of skill in the art are familiar with the implementation of such analyses, and with developing scoring or ranking systems in order to assign a weighted comparative score to candidate antigens. For example, numeric scores may be assigned for each attribute under consideration, and the antigens with the highest cumulative totals may be selected for use. In addition, other considerations may play a role in the decision making process, some of which are practical in nature (e.g. the availability of the antigen, the ease of expressing the antigen, the ease of measuring immune responses to the antigen, etc.)

The starting pool of antigens may be any or all known Mtb open reading frame (ORF) products. Those of skill in the art are familiar with sources for identifying Mtb ORF products, e.g. the “Tuberculist” website sponsored by the Pasteur Institute. In one embodiment of the invention, the antigens from which a selection may be made include but are not limited to all ORF products of M. tuberculosis H37Rv as identified in GenBank Accession #AL123456 (NC 000962).

In a preferred embodiment of the invention, the antigens that are expressed by the rBCG are selected from but are not limited to an initial group of 189 antigens presented in Table 1.

TABLE 1 List of 189 selected antigens No. Gene (name) Size NCBI Annotation [Updated annotation]^((a)) Rv0079 273 hypothetical protein Rv0101 nrp 2512 probable peptide synthase (nrp) Rv0125 pepA 355 probable serine protease pepA (MTB32A) [DegQ] Rv0170 mce1B 346 MCE - family protein mce1B [Ttg2C, periplasmic component ABC transporter] Rv0198c 663 possible zinc metalloprotease [PepO, predicted metalloendopeptidase] Rv0211 pckA 606 phosphoenolpyruvate carboxykinase pckA (GTP) Rv0227c 421 probable conserved membrane protein Rv0243 fadA2 440 acetyl-coA acetyltransferase [beta keto-thiolase] Rv0251c hsp [acr2] 159 heat-shock protein hsp (heat-stress induced ribosome-binding protein A) Rv0282 631 hypothetical protein [AAA ATPase] Rv0283 538 possible conserved membrane protein [ATP/GTP-binding protein] Rv0284 [ftsk] 1330 possible conserved membrane protein [chromosome partition ATPase] Rv0285 PE5 102 PE family protein (PE5) Rv0286 PPE4 513 PPE family protein (PPE4) Rv0287 exsG 97 ESAT-6-like protein esxG (conserved hypothetical protein TB9.8) Rv0288 esxH (TB10.4) 96 low MW protein antigen 7 esxH (10 kDa antigen) CFP-7, TB10.4) Rv0289 295 hypothetical protein [transporter] Rv0290 472 probable conserved trans-membrane protein (mgcP3) [transporter] Rv0292 331 probable conserved trans-membrane protein Rv0350 dnaK 625 molecular chaperone DnaK Rv0351 grpE 235 probable grpE protein (HSP-70 cofactor) Rv0383c 284 possible conserved secreted protein Rv0384c clpB 848 probable endopeptidase ATP binding protein ClpB (chain B) heat-shock protein F84.1 Rv0450c mmpl4 967 probable conserved trans-membrane mmpL4 [drug exporting] Rv0467 icl [aceA] 428 isocitrate lyase (icl) Rv0468 fadB2 268 3-hydroxybutyryl-CoA dehydrogenase Rv0503c cmaA2 302 cyclopropane-fatty-acyl-phospholipid-synthase 2 (cyclopropane mycolic acid synthase 2, CMAA2) Rv0569 88 hypothetical protein Rv0572c 113 hypothetical protein Rv0574c [pgsA] 380 hypothetical protein [pgsA poly-gamma-glytamyl biosyntehsis] Rv0588 yrbE2B 295 conserved hypothetical integral membrane protein YrbE2B [putative Ttg2B, ABC-type transport system involved in resistance to organic solvents, permease component) Rv0628c 383 hypothetical protein Rv0685 Tuf 396 elongation factor Tu, tuf [iron-regulated] Rv0754 PE_PGRS11 584 PE-PGRS family protein (PE_PGRS11) [phosphoglycerate mutase] Rv0798c cfp29 265 29 kDa antigen CFP-29 [linocin M-18 bacteriocin] Rv0824c desA1 389 probable acyl-[-acyl-carrier-desaturase desA1] Rv0847 lpqS 130 probable lipoprotein LPQS Rv0867c rpfA 407 possible conserved trans-membrane protein [transglycosylase, rpfA] Rv0885 340 hypothetical protein Rv1006 567 hypothetical protein Rv1009 rpfB 362 possible resuscitation-promoting factor rpfB [transglycosylase, C5 adhesion domain] Rv1057 393 hypothetical protein Rv1094 desA2 271 possible acyl-[-acyl-carrier protein] desaturase (DESA2) Rv1124 ephC 316 probable epoxide hydrolase EPHC (epoxide hydratase) Rv1130 [prpD] 526 hypothetical protein [2 methyl-citrate dehydratase] Rv1131 gltA1 393 citrate synthase (glaA1) Rv1169c PE11 100 PE family protein (PE11) [triacyl glycerol lipase] Rv1174c [sak5] 110 low MW T-cell antigen TB8.4 [secretion antigen SA5K] Rv1182 papA3 472 probable conserved polyketide synthase associated protein PAPA3 Rv1186c 538 hypothetical protein [regulator of polyketide synthase expression] Rv1187 rocA 543 probable proline-5-carboxylate dehydrogenase rocA Rv1188 329 probable proline dehydrogenase Rv1196 PPE18 391 probable proline dehydrogenase PPE family protein (mtb39a) [MTB39a] Rv1221 sigE 257 RNA polymerase sigma-70 factor (SigE) Rv1347c 210 hypothetical protein [GCN5-related N- acetyltransferase fold] Rv1348 [lrtA] 859 probable drug-transport trans-membrane ATP-binding protein ABC-transporter [MdlA/MsbA essential ABC transporter, siderophore interaction protein] Rv1349 [irtB] 579 possible drug-transport trans-membrane ATP-binding protein ABC-transporter [ATM1 ABC siderophore- iron transporter] Rv1411c lprG 236 possible conserved lipoprotein lprG Rv1436 gap 339 glyceraldehyde-3-phosphate dehydrogenase Rv1461 [sufB] 846 hypothetical protein [sufB, cytosine desulfurase activator] Rv1462 [sufD] 397 hypothetical protein [sufD, cytosine desulfurase activator] Rv1464 csd [sufS] 417 possible cysteine desulfurase csd [SufS] Rv1465 [nifU] 162 possible nitrogen fixation related protein [IscU] Rv1466 115 hypothetical protein [PaaD, predicted metal-sulfur cluster biosynthetic enzyme] Rv1477 ripA 427 hypothetical invasion protein [Nlp_p60 cell-wall hydrolase] Rv1478 241 hypothetical invasion protein [Nlp_p60 cell-wall hydrolase] Rv1594 nadA 349 quinoline synthetase (nadA) Rv1636 TB15.3 146 iron-regulated conserved hypothetical protein TB15.3 [USP] Rv1733c 210 probable conserved transmembrane protein Rv1734c 80 hypothetical protein [dihydrolysine acetyl-transferase] Rv1735c 165 hypothetical membrane protein Rv1736c narX 652 possible nitrate reductase narX Rv1737c narK2 395 possible nitrate/nitrite transporter narK2 Rv1738 94 hypothetical protein Rv1793 esxN 94 putative ESAT-6-like protein ESXN (ESAT-6-like protein 5) Rv1812c [ndH] 400 possible dehydrogenase [Ndh, NADH dehydrogenase, FAD-containing subunit] Rv1813c 143 hypothetical protein Rv1876 bfrA 159 probable bacterioferritin bfrA Rv1884c rpfC 176 probable resuscitation-promoting factor rpfC [transglycosylase] Rv1886c ftpB (Ag85B) 325 secreted antigen 85-B FBPB (85-B) (mycolyl-transferase 85B) Rv1908c katG 740 catalase-peroxidase-peroxinitritase-T katG Rv1926c mpt63 159 immunogenic protein MPT63 (16 kDa immunoprotective extracellular antigen) Rv1980c mpb64 228 immunogenic protein MPT64 Rv1986 199 probable conserved integral membrane protein [lysine efflux permease] Rv1996 317 hypothetical protein [USP] Rv1997 ctpF 905 probable metal cation transporter P-type APTase cptF Rv1998c 258 hypothetical protein Rv2004c 498 hypothetical protein [predicted kinase] Rv2005c 295 hypothetical protein [USP-like] Rv2006 otsB1 1327 probable trehalose-6-phosphate phosphatase OSTB1 Rv2007c fdxA 114 probable ferrodoxin fdxA Rv2008c 441 hypothetical protein [predicted ATPase] Rv2011c 143 hypothetical protein[transcription regulator] Rv2028c 279 hypothetical protein [USP] Rv2029c pfkB 339 possible phosphofructokinase (pfkB) Rv2030c 681 hypothetical protein [putative esterase/transferase] Rv2031c acr 144 heat-shock protein HspX (alpha-crystallin homolog) (α-crystallin) 14 kDa antigen Hsp16.3 Rv2032 acg 331 conserved hypothetical protein Acg Rv2110c [prcB] 291 proteosome (beta subunit) PrcB [HslV protease] Rv2123 PPE37 473 PPE family protein (PPE37) Rv2140c TB18.6 [pepB] 176 hypothetical protein (TB18.6) [PEBP, bacterial/archeal phosphatidylethanolamine- binding protein] Rv2182c [pslC] 247 1-acylglycerol-3-phosphate O-acyltransferase Rv2224c [caeA] 520 probable exported protease [cae, carboxylase A, TAP-like protein] Rv2244 acpM 115 acyl-carrier protein (acpM) Rv2245 kasA 416 3-oxoacyl-(acyl carrier protein) synthase (kasA) Rv2246 kasB 438 3-oxoacyl-(acyl carrier protein) synthase (kasB) Rv2251 [glcD] 475 possible flavoprotein [GlcD, FAD/FMN-containing dehydrogenases] Rv2377c mbtH 71 putative conserved protein MBTH Rv2378c mbtG 431 lysine-N-oxygenase MBTG [lucD] Rv2380c mbtE 1682 peptide synthase MBTE [EntF] Rv2381c mbtD 1004 polyketide synthase MBTD [acyl-transferase, KR domain] Rv2382c mbtC 444 polyketide synthase MBTC Rv2383c mbtB 1414 phenyloxazoline synthase (MBTB) [EntF, GrsT] Rv2386c mbtI 450 anthranilate synthase component I (MBTA) [salicylate synthase] Rv2389c rpfD 154 probable resuscitation-promoting factor rpfE [transglycosylase] Rv2428 ahpC 195 alkyl hydroxyperoxide reductase C protein ahpC Rv2429 ahpD 177 alkyl hydroxyperoxide reductase D protein ahpD Rv2430c PPE41 194 PPE family protein (PPE41) Rv2450c rpfE 172 probable resuscitation-promoting factor rpfE [transglycosylase] Rv2457c clpX 426 ATP-dependent protease ATP-binding subunit (CplX) Rv2466c 207 hypothetical protein [putative DsbA_FrnE] [thiol oxidoreductase, polyketide biosynthesis] Rv2510c 533 hypothetical protein [ATP binding domain] Rv2515c 415 hypothetical protein [putative zinc peptidase] Rv2516c 250 hypothetical protein Rv2557 224 hypothetical protein Rv2590 fadD9 1168 probable fatty-acid coA ligase fadD9 Rv2620c 141 probable conserved trans-membrane protein Rv2621c 224 possible transcriptional regulatory protein Rv2622 273 possible methyltransferase (methylase) Rv2623 TB31.7 297 hypothetical protein TB31.7 [USP] Rv2625c 393 probable conserved trans-membrane alanine-rich and leucine-rich protein [zinc-protease M-50 CBS domain] Rv2626c 143 hypothetical protein [CBS pair-binding/regul, euk] Rv2627c 413 hypothetical protein Rv2628 120 hypothetical protein Rv2629 374 hypothetical protein [peptide chain release factor erF1] Rv2657c 86 probable phiRv2 prophage protein [MerR regulatory protein] Rv2659c 375 probable phiRv2 prophage integrase Rv2660 75 hypothetical protein Rv2710 sigB 323 RNA polymerase sigma factor (SigB) Rv2744c 35kd-Ag [pspA] 270 conserved 35 kDa alanine-rich protein [phage-shock protein IM30] Rv2780 ald 371 secreted L-alanine dehydrogenase ald (40 kDa antigen, TB43) Rv2833c ugpB 436 probable Sn-glycerol-3-phosphate-binding lipoprotein UGPB Rv2856 nicT 372 possible nickel-transport integral membrane protein nicT Rv2869c 404 probable conserved trans-membrane protein [putative pdz membrane associated zinc-metalloprotease] Rv2875 mpt70 193 major secreted immunogenic protein MPT70 Rv2930 fadD26 626 fatty-acid-coA ligase FadD26 Rv2986c hupB 214 probable DNA-binding protein HU homolog HupB (histone-like protein, 21 kDa laminin-2 binding protein) Rv2999 lppY 321 probable conserved lipoprotein LPPY Rv3126c 104 hypothetical protein Rv3127 344 hypothetical protein [possible nitroreductase] Rv3129 110 hypothetical protein Rv3130c tgs1 463 hypothetical protein [diacylglycerol acyltransferase] Rv3131 nfnB 332 hypothetical protein [possible nitroreductase NfnB] Rv3132c devS 578 two-component sensor histidine kinase DevS Rv3133c devR 217 two-component transcription regulatory protein DevR Rv3134c 268 hypothetical protein [USP] Rv3139 fadE24 468 probable acyl-coA dehydrogenase FadE24 Rv3140 fadE23 401 probable acyl-coA dehydrogenase FadE23 Rv3173c 200 probable transcriptional regulatory protein (TetR/acRR family) Rv3229c desA3 427 possible linoleoyl-coA desaturase (delta-(6)-desaturase) Rv3250c rubB 495 probable rubredoxin rubB Rv3251c rubA 55 probable rubredoxin rubA Rv3283 sseA 297 probable thiosulfate sulfurtranserase SSEA (rhodanase) Rv3290c lat 449 L-lysine epsilon aminotransferase Rv3347c PPE55 3157 PPE family protein (PE55) [8 copies pentapeptide repeats] Rv3372 ostB2 391 possible trehalose-6-phosphate phosphatase (OSTB2) Rv3515c fadD19 548 AMP-dependent fatty-acid-coA ligase FadD19 Rv3516 echA19 263 enoyl-coA hydratase/isomerase (echA19) Rv3546 fadA5 391 acetyl-coA acetyltransferase (FadA5) Rv3570c [ncnH] 394 possible oxidoreductase [NcnH, naphthocyclinone hydroxylase] Rv3593 lpqF [penP] 452 probable conserved lipoprotein LPQF [PenP, beta-lactamase class A] Rv3597c lsr2 112 probable iron-regulated LSR2 protein precursor Rv3616c [espB] 392 conserved hypothetical alanine-rich and glycine-rich protein [ESAT secretion system component] Rv3619c esxV 94 putative ESAT-6-like protein ESXV (ESAT-6-like protein 1) Rv3660c 350 hypothetical protein Rv3763 lpqH 159 19 kDa lipoprotein antigen precursor LPQH Rv3804c fbpA (Ag85A) 338 secreted antigen 85-A FBPA (85-A) (mycolyl-transferase 85A) Rv3812 PE_PGRS62 504 PE-PGRS family protein (PE_PGRS62) Rv3833 263 transcriptional regulatory protein [probable araC family] Rv3839 258 hypothetical protein Rv3840 137 probable transcriptional regulatory protein [cell-envelope related transcription attenuator] Rv3841 bfrB 181 possible bacterioferritin bfrB Rv3871 [Ftsk] 591 hypothetical protein [FtsK_SPOIIIE] Rv3873 PPE68 368 PPE family protein [PPE68, RD1 T/B immunogen] Rv3874 esxB 100 10 kDa culture filtrate antigen ESXB (LHP, CFP-10) Rv3875 esxA 95 6 kDa early secretory antigenic target ESXA (ESAT-6) Rv3876 [Ftsk] 666 conserved hypothetical proline and alanine-rich protein [chromosome partitioning ATPase] Rv3878 280 conserved hypothetical alanine-rich protein Rv3879c 729 hypothetical alanine and proline-rich protein ^((a))NCBI annotation is based on Accession # AL123456 (NC_000962); updated annotation is based on bioinformatic analyses, data from MTB-related servers and experimental evidence.

In a more preferred embodiment of the invention, the antigens that are expressed by the rBCG are the 45 antigens presented in Tables 2 and 3.

TABLE 2 Top-ranking antigens (according to Class/Phase) Gene NCBI annotation Score Score No. (name) Size [Updated annotation]^((a)) Qual Quant Class: DORMANCY/DosR Rv1738 94 hypothetical protein 9 14 Rv2623 TB31.7 297 heat-shock protein TB31.7 9 14 [universal stress protein] Rv2031c acr 144 heat-shock protein HspX (alpha- 8 14 (α-crystalllin) crystallin homolog) 14 kDa antigen Hsp16.3 Rv2032 acg 331 conserved hypothetical protein Acg 8 13 [nitroreductase] Rv2626c 143 hypothetical protein 8 13 [CBS pair-binding/regulation, euk] Rv2005c 295 hypothetical protein 8 12 [USP-like] Rv3127 344 hypothetical protein 8 12 [possible nitroreductase] Rv1733c 210 probable conserved 8 11 trans-membrane protein Rv1996 317 hypothetical protein [USP] 8 10 Rv2628 120 hypothetical protein 8 9 Rv0079 273 hypothetical protein 7 11 Rv3130c [tgs1] 463 hypothetical protein 7 11 [diacylglycerol acyltransferase] Rv3131 [nfnB] 332 hypothetical protein 7 11 [possible nitroreductase NfnB] Rv1813c 143 hypothetical protein 7 9 Rv2006 otsB1 1327 probable trehalose-6-phosphate 7 9 phosphatase OTSB1 Rv2029c pfkB 339 possible phophofructokinase (pfkB) 7 9 Rv2627c 413 hypothetical protein 7 9 [serine endopeptidase] Rv2030c 681 hypothetical protein 6 10 [putative esterase/transferase] Rv3132c devs 578 two component sensor histidine 6 10 kinase DEVS Rv2629 374 hypothetical protein 6 9 [peptide release factor erf-1] Class: RESUSCITATION Rv2450c rpfE 172 probable resuscitation-promoting 9 14 factor rpfE [transglycosylase] Rv1009 rpfB 362 possible resuscitation-promoting 9 13 factor rpfB [transglycosylase, C5 adhesion domain] Rv0867c rpfA 407 possible conserved trans-membrane 9 12 protein [transglycosylase, rpfA] Rv2389c rpfD 154 probable resuscitation-promoting 8 10 factor rpfD [transglycosylase] Rv1884c rpfC 176 probable resuscitation-promoting 7 8 factor rpfC [transglycosylase] Class: REACTIVATION Rv1009 rpfB 362 possible resuscitation-promoting 9 13 factor rpfB [transglycosylase, C5 adhesion domain] Rv0867c rpfA 407 possible conserved trans-membrane 9 12 protein [transglycosylase, rpfA] Rv0288 esxH (TB10.4) 96 low MW protein antigen 7 esxH 8 13 (10 kDa antigen) CFP-7, TB10.4) Rv0685 Tuf 396 elongation factor Tu 8 9 [iron-regulated] Rv0824c desA1 389 probable acyl[-acyl-carrier- 7 10 desaturase desA1] Rv2744c 35kd-Ag 270 conserved 35 kDa alanine-rich 7 8 [pspA] protein [phage-shock protein IM30] Rv3347c PPE55 3157 PPE 55 Family Protein 6 10 [8 copies pentapeptide repeats] Rv1130 prpD 526 hypothetical protein 6 9 [2 methyl-citrate dehydratase] Rv1169c PE11 100 PE family protein (PE11) 6 9 [triacyl glycerol lipase] Class: CLASSICAL Rv1886c fbpB (Ag85B) 325 mycolyl transferase/fibronectin 8 14 binding Rv1980c mpb64 228 antigen MPT64/MPB64 7 13 Rv3804c fbpA (Ag85A) 338 mycolyl transferase/fibronectin 7 13 binding Rv3875 esxA 95 6 kDa early secretory antigen esxA 6 11 Rv1926c mpt63 159 immunogenic protein MPT63 6 10 (16 kDa immunoprotective extracellular protein) Rv0467 icl 428 isocitrate lyase (icl) [AceA] 6 9 Class: OTHERS Rv3873 PPE68 368 hypothetical protein 8 13 Rv1908c katG 740 catalase-peroxidase-peroxinitritase-T 7 10 (KATG heme dependent) Rv1174c sak5 110 low MW T-cell antigen TB8.4 7 9 (secreted) Rv1349 irtB 579 probable drug transport ATP-binding 7 9 protein ABC transporter [ATM1 ABC siderophor-iron transporter] Rv2780 ald 371 secreted L-alanine dehydrogenase 7 9 ALD (40 kDa antigen) (TB4.3, cell associated pyridine nucleotide transhydrogenase) Rv2620c 141 probable conserved transmembrane 7 8 protein Rv1793 esxN 94 putative ESAT-6 like protein 6 9 (ESXN, ESAT-6 like protein 5) ^((a))NCBI annotation is based on Accession # AL123456 (NC_000962); updated annotation is based on bioinformatic analyses, data from MTB-related servers and experimental evidence. The list of 45 high-ranking antigens were classified according to the following classes:

-   -   DosR regulon genes (Voskuil et al., 2003, J. Exp Med         198(5):705-713; Voskuil et al. 2004, Tuberculosis 84, 218-227)     -   Resuscitation genes     -   Reactivation genes (essentially according to Talaat et al.,         2007, Proc. Natl. Acad. Sci. 189(21):7877-7886)     -   Classical genes     -   Others (mainly involved in persistence and stress response)         Some of the antigens appear in more than one class in Table 3.

TABLE 3 Top-ranking antigens sorted by qualitative and quantitative scores NCBI Annotation Score Score No. Gene (Name) Size [Updated annotation]^((a)) (Qual) (Quant) Class/Phase GROUP 1 Rv1738 94 hypothetical protein 9 14 DosR Rv2450c rpfE 172 probable resuscitation- 9 14 Resuscitation promoting factor rpfE [transglycosylase] Rv2623 TB31.7 297 hypothetical protein 9 14 DosR TB31.7 [USP] Rv1009 rpfB 362 possible resuscitation- 9 13 Reactivation promoting factor rpfB Resuscitation [transglycosylase, C5 adhesion domain] Rv0867c rpfA 407 possible conserved 9 12 Reactivation trans-membrane protein Resuscitation [transglycosylase, rpfA] Rv2031c acr 144 heat-shock protein HspX 8 14 DosR (α-crystallin) (α-crystallin homolog) 14 kDa antigen Hsp 16.3 Rv1886c fbpB 325 secreted antigen 85-B 8 14 Classical (Ag85B) FBPB (85-B) (mycolyl-transferase 85B) Rv0288 esxH 96 low MW protein antigen 7 8 13 Reactivation (TB10.4) esxH (10 kDa antigen) CFP-7, TB10.4) Rv2032 acg 331 conserved hypothetical 8 13 DosR protein Acg [nitroreductase] Rv2626c 143 hypothetical protein 8 13 DosR [CBS pair- binding/regulation, euk] Rv3873 PPE68 368 PPE family protein [PPE68, 8 13 Others RD1 T/B immunogen] Rv2005c 295 hypothetical protein 8 12 DosR [USP-like] Rv3127 344 hypothetical protein 8 12 DosR [possible nitroreductase] GROUP II Rv1733c 210 probable conserved 8 11 DosR trans-membrane protein Rv1996 317 hypothetical protein 8 10 DosR [USP] Rv2389c rpfD 154 probable resuscitation- 8 10 Resuscitation promoting factor rpfD [transglycosylase] Rv0685 Tuf 396 elongation factor Tu 8 9 Reactivation [iron-regulated] Rv2628 120 hypothetical protein 8 9 DosR Rv1980c mpb64 228 immunogenic protein 7 13 Classical MPT64 Rv3804c fbpA 338 secreted antigen 85-A 7 13 Classical (Ag85A) FBPA (85-A) (mycolyl-transferase 85A) Rv0079 273 hypothetical protein 7 11 DosR Rv3130c [tgs1] 463 hypothetical protein 7 11 DosR [diacylglycerol acyltransferase] Rv3131 [nfnB] 332 hypothetical protein 7 11 DosR [possible nitroreductase NfnB] Rv0824c desA1 389 probable acyl[-acyl-carrier- 7 10 Reactivation desaturase desA1] Rv1908c katG 740 catalase-peroxidase- 7 10 Others peroxinitritase-T katG Rv1174c [sak5] 110 Low Mw T-cell antigen 7 9 Others TB8.4 [secretion antigen SA5K] Rv1349 [irtB] 579 probable drug transport 7 9 Others ATP-binding protein ABC transporter [ATM1 ABC siderophore-iron transporter] Rv1813c 143 hypothetical protein 7 9 DosR Rv2006 otsB1 1327 probable trehalose-6- 7 9 DosR phosphate phosphatase OSTB1 Rv2029c pfkB 339 possible 7 9 DosR phosphofructokinase (pfkB) Rv2627c 413 hypothetical protein 7 9 DosR [serine endopeptidase] Rv2780 ald 371 secreted L-alanine 7 9 Others dehydrogenase ald (40 kDa antigen, TB43) GROUP III Rv1884c rpfC 176 probable resuscitation- 7 8 Resuscitation promoting factor rpfC [transglycosylase] Rv2620c 141 probable conserved 7 8 Others transmembrane protein Rv2744c 35kd-Ag 270 conserved 35 kDa alanine- 7 8 Reactivation [pspA] rich protein [phage-shock protein IM30] Rv3875 esxA 95 6 kDA early secretory 6 11 Classical antigenic target ESXA (ESAT-6) Rv1926c mpt63 159 immunogenic protein 6 10 Classical MPT63 (16 kDa immunoprotective extracellular protein) Rv2030c 681 hypothetical protein 6 10 DosR [putative esterase/transferase] Rv3132c devs 578 two component sensor 6 10 DosR histidine kinase DEVS Rv3347c PPE55 3157 PPE family protein (PE55) 6 10 Reactivation [8 copies pentapeptide repeats] Rv0467 icl 428 isocitrate lyase(icl) [AceA] 6 9 Classical Rv1130 [prpD] 526 hypothetical protein 6 9 Reactivation [2 methyl-citrate dehydratase] Rv1169c PE11 100 PE family protein (PE11) 6 9 Reactivation [triacyl glycerol lipase] Rv1793 esxN 94 putative ESAT-6-like 6 9 Others protein ESXN (ESAT-6-like protein 5) Rv2629 374 hypothetical protein 6 9 DosR [peptide release factor erF1] ^((a))NCBI annotation is based on Accession # AL123456 (NC_000962); updated annotation is based on bioinformatic analyses, data from MTB-related servers and experimental evidence.

The list of 45 high-ranking antigens were sorted by the qualitative score and then by the quantitative score according to this invention and as discussed in Example 1. This method leads to 3 groups as follows:

-   Group I: antigens with qualitative scores 9-8, and within the     antigens with a qualitative score of 8, the quantitative score     cutoff is 12. -   Group II: antigens with qualitative scores 8-7, the quantitative     score cutoff of 7 is 9. -   Group III: antigens with qualitative score 7-6, the quantitative     score cutoff of 6 is 9.     Antigens with more than 5 transmembrane segments were removed from     the list. B1

In general, at least one such antigen will be overexpressed and several different antigens may be overexpressed. For example, about 1-20 or more of such antigens, or alternatively about 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or only 1 of such antigens will be overexpressed by the rBCG. Further, multiple copies of one or more of the antigens may be encoded and overexpressed in the rBCG. The amino acid sequences of selected antigens, and the nucleic acid sequences that encode them, are depicted in FIGS. 5A-B to 24A-B.

In addition, the rBCG contains nucleic acid sequences comprising one or more genes that encodes and overexpresses at least one Mtb resuscitation/reactivation antigen. Those of skill in the art will be aware that the precise definition of “resuscitation” and “reactivation” antigens may vary somewhat within the field, and in some cases, the definitions may overlap. For the purposes of the invention, it is not necessary to separate resuscitation and reactivation antigens as all are identified as being of significance in the outgrowth of Mtb from the latent state. In the most accurate sense, resuscitation antigens (i.e. resuscitation promoting factors) are a subset of reactivation antigens defined as having significant sequence or functional homology to the resuscitation promoting factor of Micrococcus luteus (G Mukamolova et al, Archives of Microbiology, Volume 172, 1999). For the purposes of the present application, a “reactivation” antigen is a protein expressed by M. tuberculosis which elicits an immune response in humans with active tuberculosis but not latent Mtb infection. They may also be identified as immunogens expressed by Mtb during outgrowth from a non-replicative stationary phase in the Wayne model of latent tuberculosis. This may include molecules that are expressed during the emergence of Mtb from the dormant latent state into active tubercle bacilli. Examples of suitable reactivation/resuscitation antigens include Rv0867c, Rv0288, Rv1009, Rv0685, Rv0824c, Rv2744c, Rv3347c, Rv1130, Rv1169c, Rv1009, Rv1884c, Rv2389c, and Rv2450c. In a preferred embodiment, the reactivation/resuscitation antigens expressed are Rv0867c, Rv1884c, and Rv2389c.

In addition, in the rBCG of the invention the genes of the DosR (Dormancy Survival Regulator) regulon, or a portion thereof, are upregulated and expressed. The entire regulon may be upregulated, or a suitable portion thereof. For example, genes that encode antigens that are recognized by individuals with latent TB may be the most suitable for upregulation. Examples of DosR upregulated antigens include Rv1738, Rv2623, Rv2031c, Rv2032, Rv2626c, Rv2005c, Rv3127, Rv1733c, Rv1996, Rv2628, Rv0079, Rv3130c, Rv3131, Rv1813c, Rv2006, Rv2029c, Rv2627c, Rv2030c, Rv3132c, and Rv2629. It is noteworthy that some overlap exists between latency and reactivation antigens, possibly reflecting the extended presence of latency related antigens in previously dormant organisms re-entering an active growth phase or their function in both the dormant and actively replicating state after reactivation in the mammalian host. Antigens that may be considered to overlap between latency and reactivation are listed in Table 4.

TABLE 4 Antigens that overlap latency and reactivation Reactivation/dosR Antigens Rv1996 Rv2005 Rv2029 Rv2623 Rv2626 Rv2727

By “up-regulate” we mean that expression of each of the individual genes of the regulon or their translated proteins is increased above the level at which they are expressed when the regulon is in a “repressed” state. Proteins of the DosR regulon are normally expressed at a relatively low level. Upon oxygen starvation and/or the presence of oxidative nitrogen compounds the DosS and DosT proteins of TB complex organisms autophosphorylate and transfer this phosphate to DosR. DosR then binds to discrete sequences upstream of DosR regulated genes thereby activating their transcription and upregulating this group of genes and gene products which constitute the DosR regulon. Upregulation within the practice of this invention mimics this oxygen starvation effect where the DosR genes have increased transcription.

Those of skill in the art are familiar with approaches to genetically engineering an organism in order to up-regulate selected genes of interest, or selected regulons of interest. Such approaches include but are not limited to overexpression of the regulator, introduction of mutations in the regulator or sensor which render them constitutively active, the introduction of regulators which mimic the function of the regulator in question, introduction of kinases or feedback loop products which activate the sensor or regulator, or the introduction of genes/gene products which mimic the environmental state which causes activation of the sensor or regulator. In a preferred embodiment, the DosR regulon is up-regulated by over expressing the response regulator DosR (Rv3133c) of the DosRST “two component” regulatory system.

In another preferred embodiment, the vaccine includes one or more of Rv1908, Rv3873, Rv2780 and Rv1349. These are immunopotent antigens which were identified in silico and/or by experimentation.

In addition, the rBCG of the invention may encode antigens selected based on other criteria, such as demonstrated protective efficacy in an animal model or the expression of the antigen by Mtb but not BCG (J Mattow et al., Electrophoresis, 22:2936-2946, 2001, P. R. Jungblut, Molecular Microbiology, 33:1103-1117, 1999, H. J. Mollenkopf et al, Infection and Immunity, 72:6471-6479, 2004). In a preferred embodiment, the rBCG of the invention expresses Rv3407, which is normally expressed by Mtb but not BCG and has been shown to protect against tuberculosis in a mouse model.

The BCG that is genetically engineered as described herein may be of any BCG strain considered suitable, including but not limited to BCG strains BCG₁₃₃₁, BCG Pasteur, BCG Tokyo, BCG Copenhagen, BCG Moreau, or BCG Moscow.

In a preferred embodiment, the strain is BCG₁₃₃₁. In addition, the rBCG may be further genetically engineered to possess other traits, for example: a perfringolysin O (pfo) gene (in order to facilitate escape from the endosome); to express various selection markers such as antibiotic resistance or an auxotrophic selection marker in which a gene critical to the rBCG (e.g. for leucine or lysine synthesis) is deleted and must be complemented (e.g. by an extrachromsomal element that encodes the missing crucial gene) in order for the bacterium to survive; by deletion of genes or inhibition of the function of gene products which suppress apoptosis, etc.

In general, the rBCG of the invention is genetically engineered to overexpress selected antigens by introduction of genes encoding the antigens of interest into the chromosome of the rBCG under the transcriptional control of highly active expression control sequences, which may include those which are most active during mammalian infection. However, the genes encoding the antigens of interest could also be expressed from an extrachromosomal plasmid under the transcriptional control of highly active expression control sequences. Expression control sequences include but are not limited to promoters, ribosomal entry sites, etc.

The present invention further provides compositions for use in eliciting an immune response in and/or vaccinating a mammal. The compositions may be utilized as a vaccine against Mtb. The compositions of the invention include genetically engineered rBCG as described herein, and a pharmacologically suitable carrier. The preparation of such compositions for use as vaccines is well known to those of skill in the art. Typically, such compositions are prepared either as liquid solutions or suspensions, however solid forms such as tablets, pills, powders and the like are also contemplated. Solid forms suitable for solution in, or suspension in, liquids prior to administration may also be prepared. The preparation may also be emulsified. The active ingredients may be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredients. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol and the like, or combinations thereof. In addition, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and the like. In addition, the composition may contain other adjuvants. If it is desired to administer an oral form of the composition, various thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders and the like may be added. The composition of the present invention may contain any such additional ingredients so as to provide the composition in a form suitable for administration. The final amount of rBCG in the formulations may vary. However, in general, the amount in the formulations will be from about 1-99%. The compositions may further comprise additional adjuvants, suitable examples of which include but are not limited to Seppic, Quil A, Alhydrogel, etc.

Vaccine formulation also involves studies to determine maximum bacterial viability and stability throughout the manufacturing process. This includes determination of maximum organism viability (live to dead) during culture utilizing a variety of commonly used medium for the culture of Mycobacteria to include the addition of glycerol, sugars, amino acids, and detergents or salts. After culture cells are harvested by centrifugation or tangential flow filtration and resuspended in a stabilizing medium that allows for protection of cells during freezing or freeze-drying process. Commonly used stabilizing agents include sodium glutamate, amino acids or amino acid derivatives, glycerol, sugars or commonly used salts. The final formulation will provide sufficient viable organisms to be delivered by intradermal, percutaneous injection, perfusion or oral delivery with sufficient stability to maintain and adequate shelf life for distribution and use.

The methods of the present invention involve administering a composition comprising the rBCG of the invention in a pharmacologically acceptable carrier to a subject, usually a human mammal. The vaccine preparations of the present invention may be administered by any of the many suitable means which are well known to those of skill in the art, including but not limited to by injection, orally, intranasally, by ingestion of a food product containing the antigen, etc. However, in a preferred embodiment, the mode of administration is intradermal injection. In addition, the compositions may be administered alone or in combination with other medicaments or immunogenic compositions, e.g. as part of a multi-component vaccine. Further, administration may be a single event, or multiple booster doses may be administered at various timed intervals to augment the immune response. In one embodiment, the vaccine preparation of the invention is used for an initial immunization, and the individual receiving the initial vaccine is then “boosted” with one or more different vaccine compositions, e.g. a known attenuated BCG vaccine. Alternatively, an individual may be vaccinated with another vaccine preparation and boosted one or more times with the vaccine preparation of the present invention. In some embodiments of the invention, the boosting compositions include nucleic acids encoding one or more Mtb antigens, for example: i) one or more antigens such as Rv1738, Rv2623, Rv2031c, Rv2032, Rv2626c, Rv2005c, Rv3127, Rv1733c, Rv1996, Rv2628, Rv0079, Rv3130c, Rv3131, Rv1813c, Rv2006, Rv2029c, Rv2627c, Rv2030c, Rv2629, Rv2450c, Rv1009, Rv0867c, Rv2389c, Rv1884c, Rv0288, Rv0685, Rv0824c, Rv2744, Rv3347c, Rv130, Rv1169c, Rv1886, Rv1980c, Rv3804c, Rv3875, Rv1926c, Rv0467, Rv3873, Rv1908c, Rv1174c, Rv2780, Rv2620c, Rv1793, Rv1349 and Rv3132; ii) one or more antigens such as Rv1996, Rv2005, Rv2029, Rv2623, Rv2626 and Rv2727; iii) one or more antigens such as Rv2626, Rv1738, Rv2623, Rv1733, Rv2032, Rv3131, Rv3127, Rv3130c, Rv3804c and Rv1886c; a or more antigens including at least one of a dosR antigen, a reactivation antigen and/or a resuscitation antigen e.g., one antigen from each stage (latency, reactivation and resuscitation) of the life cycle of M. tuberculosis.

A particular advantage of the vaccine preparation of the present invention is that administration may be prophylactic, i.e. before exposure to the bacteria has occurred, or is suspected to have occurred, or after the fact, i.e. after a known or suspected exposure, or therapeutically, e.g. after the occurrence of disease symptoms associated with bacterial infection. This is because antigens that are involved in Mtb life cycle or infection and disease processes such as resuscitation and reactivation are included in the vaccine. Thus, the vaccine is useful not only for preventing the initial establishment of an Mtb infection, but also for preventing the reactivation of a latent Mtb infection.

Prior to administration to humans as a vaccine, the genetically engineered rBCG strains of the present are tested according to methods that are well-known to those of skill in the art. For example, tests for toxicity, virulence, safety, etc. are carried out in suitable animal models, e.g. in mice, guinea pigs, etc., some of which are immunocompromised. The ability of the vaccine preparations to elicit an immune response is likewise typically tested in suitable animal models, e.g. mice, guinea pigs, etc. In addition, protection studies involving vaccination, boosting, and subsequent challenge with live Mtb may be carried out using suitable animal models such as mice, guinea pigs, and non-human primates. Finally, those of skill in the art are familiar with the arrangements for carrying out clinical trials in consenting humans, in order to test the efficacy of the vaccine preparations. For details, see, for example, United States patent application 20060121054 (Sun et al.) published Jun. 8, 2006, and references cited therein.

By “eliciting an immune response” we mean that administration of the vaccine preparation of the present invention causes the synthesis of specific antibodies (at a titer in the range of 1 to 1×10⁶, preferably 1×10³, more preferable in the range of about 1×10³ to about 1×10⁶, and most preferably greater than 1×10⁶) and/or cellular proliferation, as measured, e.g. via cellular assays in which IFN-γ production is assessed, for example, by ³H thymidine incorporation, or other suitable means. In a preferred embodiment, the immune response is a protective immune response, i.e. the immune response protects the vaccinated individual from future challenge with Mtb. However, those of skill in the art will recognize that a vaccine or immunostimulating preparation need not provide total protection in order to convey some benefit to a patient. For example, the preparations may elicit immune responses that slow or lessen the progress of disease symptoms, without fully eradicating them.

The following non-limiting Examples serve to illustrate the practice of this invention.

EXAMPLES Example 1 Selection of Suitable Antigens and Design of rBCG Based on this Selection an Initial Group of Mtb Proteins (189 Antigens, see Table 1) was Selected from all Possible 3989 Mtb ORFs, According to the Following Selection Procedure

-   (1) Compilation of available data for all 3989 TB ORF products:     Literature scan for global analyses (e.g. up/down regulation of     expression under various conditions such as hypoxia, dormancy,     interaction with macrophage, location and persistence in lung     tissue; iron regulation; transcriptomics and proteomics profiles;     mutation leading to attenuation of virulence; potential high immune     response etc.). -   (2) Establishing a subset of genes by cross matching the accumulated     data: Selection of antigens that demonstrate positive evidence in     any two independent studies originating from different criteria     mentioned above. -   (3) Iterative trimming of the above subset, aiming at selecting an     initial group of candidates (Table 1, 189 antigens), based on the     extent of effect documented in the individual studies. -   (4) Conducting bioinformatic studies for:     -   a) Further characterization of the initial group of 189         candidates by domain analyses, prediction of cellular         localization, inspection of genome context, identification of         repeat proteins, and assignment of putative function for the         unknown/hypothetical Ag (sequence similarity searches by blast         against the nr database, NCBI, domain/motifs databases, and         Mtb-related servers and databases: TB-sgc—The TB Structural         Genomics Consortium (Web site located at         www.doe-mbi.ucla.edu/TB/), Tuberculist—the database on         Mycobacterium tuberculosis genetics (Web site located at         genolist.pasteur.fr/TubercuList/), TBDB—an integrated platform         for TB drug discovery (Web site located at www.tbdb.org)         MTBreg—The Database of Conditionally Regulated Proteins in         Mycobacterium tuberculosis (Web site located at         www.doe-mbi.ucla.edu/Services/MTBreg/) and the BioHealthBase         server (Web site located at www.biohealthbase.org/GSearch).     -   b) Immunoinformatic analysis for prediction of human leukocyte         antigen (HLA)-binding targets (by NetCTL) and experimentally         documented T-cell epitopes by database (IEDB) and literature         searches. Implementing various measures for the determination of         epitopes as putative strong binders, and for the selection of         most potent candidates according to the predictions (i.e.         thresholds, and number of supertypes/population coverage). -   (5) In depth literature search for data pertaining to aspects of     virulence and vaccine development, for each of the 189 antigens     (Table 1) and/or their orthologs. -   (6) Setting up a knowledge dataset of the 189 candidates (Table 1),     by integrating all literature and data analyses (steps 4&5 above).     The selected antigens in the initial group presented in Table 1,     include known vaccine candidates, and proteins known to be: involved     in various stages of the life cycle of Mtb (dormancy, reactivation,     resuscitation); tissue specific antigens; antigens affected by     starvation; and, antigens originating in genomic sequences present     in a virulent Mtb strain.

The 189 candidate antigens were ranked in order of likely importance by the following 14 analyses:

-   1) macrophage survival/persistence; -   2) up-regulation of expression by the two-component system MprAB; -   3) response to hypoxia; -   4) involvement in reactivation; -   5) involvement in dormancy; -   6) expression in lung tissue; -   7) co-regulation with Rv2031 (Acr); -   8) whether or not the protein is secreted; -   9) the ability to act as a B-cell immunogen; -   10) iron-regulated genes; -   11) cell wall association or cell wall biogenesis; -   12) existing vaccine efficacy data; -   13) the presence of repeat domains; -   14) T-cell response, determined by a) known experimental evidence     (e.g. from the literature) and b) a determination that the antigen     likely contains one or more T-cell epitopes as assessed by the     immunoinformatics program of the Technical University of Denmark     (cbs.dtu.dk/services/NetCTL);

The presence or absence of each of these traits was scored for each of the 189 genes and a qualitative score was determined and employed as a measure to rank the list of 189 antigens and choose for the 45 best hits.

The 45 candidates were then further ranked using the same 13 criteria, by assigning internal numerical scores to each of the criteria, according to the intensity of the results and/or relevance to vaccine development. A list of the top-ranking 45 antigens is given in Table 2, along with their subgrouping according to the class/phase of infection (latency/dormancy, resuscitation/reactivation, classical and others); within the classes, the antigens are sorted by their scores. Table 3 presents the 45 antigens prioritization into 3 subset groups according to their quantitative, and subsequently—qualitative scores.

Based on this analysis, final selections of groups of antigens for use in the rBCGs were made, usually based on the antigens with the highest overall scores. In addition, for the final selection, antigens were grouped according to “type” in that the rBCG includes at least

-   1) one or more Mtb antigens, including the so-called “classical” Mtb     antigens such as 85A, 85B and TB 10.4; and -   2) at least one Mtb resuscitation/reactivation antigen selected from     Rv0867c, Rv1009, Rv1884c, Rv2389c, Rv2450c, Rv0867c, Rv0288, Rv1009,     Rv0685, Rv0824c, Rv1349, Rv2744c, Rv3347c, Rv1130, and Rv1169c.

In addition, antigens were selected based on other criteria such as demonstrated protective efficacy in an animal model, the expression of the antigen by Mtb but not BCG, or diminished expression of the antigen in BCG (J Mattow et al., Electrophoresis, 22:2936-2946, 2001, P. R. Jungblut, Molecular Microbiology, 33:1103-1117, 1999, H. J. Mollenkopf et al, Infection and Immunity, 72:6471-6479). Such Mtb-specific antigens include Rv1511, Rv2520c, Rv3407, Rv2802c and Rv3710.

Preferred combinations of antigens to be expressed in an rBCG include the following:

-   1) Classical antigens Rv1886c, Rv3804c; -   2) Resuscitation and Reactivation antigens Rv0867c, Rv1884c,     Rv2389c; and -   3) Mtb-specific antigen Rv3407.

Example 2 Construction of a Recombinant BCG Genetically Engineered to Express at Least One Classical Mtb Antigen, at Least One Mtb Resuscitation/Reactivation Antigen and in which the DosR Regulon is Upregulated

Materials and Methods: General

For the construction of an rBCG described in the following sections, restriction endonucleases (herein “REs”); New England Biolabs Beverly, Mass.), T4 DNA ligase (New England Biolabs, Beverly, Mass.) and Taq polymerase (Invitrogen, Carlsbad, Calif.) were used according to the manufacturers' protocols; Plasmid DNA was prepared using small-scale (Qiagen Miniprep® kit, Santa Clara, Calif.) or large-scale (Qiagen Maxiprep® kit, Santa Clara, Calif.) plasmids DNA purification kits according to the manufacturer's protocols (Qiagen, Santa Clara, Calif.); Nuclease-free, molecular biology grade Milli-Q water, Tris-HCl (pH 7.5), EDTA pH 8.0, 1M MgCl⁻², 100% (v/v) ethanol, ultra-pure agarose, and agarose gel electrophoresis buffer were purchased from Invitrogen, Carlsbad, Calif. RE digestions, PCRs, DNA ligation reactions and agarose gel electrophoresis were conducted according to well-known procedures (Sambrook, et al., Molecular Cloning: A Laboratory Manual. 1, 2, 3; 1989); (Straus, et al., Proc Natl Acad Sci USA. March; 87(5): 1889-93; 1990). Nucleotide sequencing to verify the DNA sequence of each recombinant plasmid described in the following sections was accomplished by conventional automated DNA sequencing techniques using an Applied Biosystems automated sequencer, model 373A.

PCR primers were purchased from commercial vendors such as Sigma (St. Louis, Mo.) or synthesized using an Applied Biosystems DNA synthesizer (model 373A). PCR primers were used at a concentration of 150-250 μM and annealing temperatures for the PCR reactions were determined using Clone manager software version 4.1 (Scientific and Educational Software Inc., Durham, N.C.). PCRs were conducted in a BioRad thermocycler (BioRad, Hercules, Calif.). The PCR primers for the amplifications were designed using Clone Manager® software version 4.1 (Scientific and Educational Software Inc., Durham N.C.). The RE digestions and the PCRs were subsequently analyzed by agarose gel electrophoresis using standard procedures (Straus et al, supra 1990; and Sambrook et al., supra 1989). A positive clone is defined as one that displays the appropriate RE pattern and/or PCR pattern. Plasmids identified through this procedure were further evaluated using standard DNA sequencing procedures, as described above.

Escherichia coli strains, such as DH5α and Stable2^(R), were purchased from Invitrogen (Carlsbad, Calif.) and served as initial host of the recombinant plasmids. Recombinant plasmids were introduced into E. coli strains by electroporation using a high-voltage eletropulse device, such as the Gene Pulser (BioRad Laboratories, Hercules, Calif.), set at 100-200Ω, 15-25 μF and 1.0-2.5 kV, as described (Straus et al, supra 1990). Optimal electroporation conditions were identified by determining settings that resulted in maximum transformation rates per mcg DNA per bacterium.

E. coli strains are typically grown on tryptic soy agar (Difco, Detroit, Mich.) or in tryptic soy broth (Difco, Detroit, Mich.), which was made according to the manufacturer's directions. Unless stated otherwise, all bacteria were grown at 37° C. in 5% (v/v) CO₂ with gentle agitation. When appropriate, the media was supplemented with antibiotics (Sigma, St. Louis, Mo.). Bacterial strains were typically stored at −80° C. suspended in (Difco) containing 30% (v/v) glycerol (Sigma, St. Louis, Mo.) at ca. 109 colony-forming units (herein referred to as “cfu”) per ml.

Mycobacterial strains were cultured in liquid media, such as Middlebrook 7H9 or Saulton Synthetic Medium, preferably at 37° C. The strains can be maintained as static or agitated cultures. In addition, the growth rate of BCG can be enhanced by the addition of oleic acid (0.06% v/v; Research Diagnostics Cat. No. 01257) and detergents such as Tyloxapol (0.05% v/v; Research Diagnostics Cat. No. 70400). The purity of BCG cultures can be evaluated by evenly spreading 100 mcl aliquots of the BCG culture serially diluted (e.g. 10-fold steps from Neat—10⁻⁸) in phosphate buffered saline (herein referred to PBS) onto 3.5 inch plates containing 25-30 ml of solid media, such as Middlebrook 7H10. In addition, the purity of the culture can be further assessed using commercially available medium such as thioglycolate medium (Science Lab, catalogue number 1891) and soybean-casein medium (BD, catalogue number 211768).

In order to insert the desired antigen expression cassettes into the chromosome of the perfringolysin expressing BCG1331 derivative described elsewhere, a plasmid was designed in silico and synthesized by DNA2.0 (Menlo Park, Calif.). The salient features of this vector (pJFINT) include an E. coli colE1 origin of replication, 3 multiple cloning sites separated by transcriptional terminators rrnBT1, T2 of pEX18gm, and rnhA, the attP phage integration region of bacteriophage L5 and the integrase gene of bacteriophage L5 (GenBank #Z18946). Immediately upstream of the L5 sequence, a selectable marker cassette consisting of a kanamycin resistance allele aphA from Tn10 (GenBank #AAM97345) and a sacB gene (Genbank # NP_(—)391325) were included. This marker cassette was flanked by direct repeats of the γΔ resolvase binding site from transposon Tn1000. This plasmid is incapable of replication in mycobacterial species and the L5 attP sequence allows for high frequency recombination with the attB region of mycobacterial chromosomes to facilitate integration of the plasmid sequence into the chromosome. The marker cassette can then be removed from the chromosome of the integrant by the introduction of γΔ resolvase and selection of markerless strains on solid media containing 10% sucrose.

An antigen expression cassette was designed in silico to encode Rv0867c, Rv1884c, and Rv2389c separated by optimized ribosomal sites under the transcriptional control of the hsp60 promoter of Mycobacterium bovis and synthesized by DNA2.0 (Carlsbad Calif.). A second expression cassette was similarly designed and constructed to encode Rv1886c, Rv3804c and Rv3407c under transcriptional control of the hsp60 promoter. Finally, a third cassette encoding Rv3133c (DosR) under the transcriptional control of the hsp60 promoter was designed and constructed. These three antigen expression cassettes were ligated into pJFINT such that each was separated by a transcriptional terminator (FIG. 2). The resulting plasmid, pRC108, was electroporated into E. coli Stable2 and the plasmid sequence of a kanamycin resistant clone was verified.

The pRC108 plasmid was isolated from a 100 ml E. coli culture and electroporated into a pfo expressing derivative of BCG Danish 1331. After electroporation, the cells were cultured overnight in 7H9 medium with 10% (v/v) OADC and 0.05% (v/v) of Tyloxapol supplementation and plated on 7H10 agar containing 50 ug/ml of kanamycin. As the plasmid does not encode a mycobacterial origin of replication, kanamycin resistance in all colonies tested was conferred by integration of the plasmid into the attB site of the BCG genome. Individual colonies were picked for PCR analysis and inoculated into 7H9 medium with 10% (v/v) OADC and 0.05% (v/v) tyloxapol for analysis of antigen expression. PCR characterization of the kanamycin resistant colonies demonstrated the presence of the entire plasmid sequence in the chromosome of the recombinant BCG, designated AFV-102pRC108. AFV-102pRC108 cultures were washed with 7H9 and used to inoculate protein-free 7H9tyloxapol cultures. Supernatants of the AFV-102pRC108 cultures were harvested by centrifugation and immunoblotted with rabbit polyclonal antisera to the transglycolase domain of the Rpf protein. Rv1009, which is cross reactive with the transglycosylase domains of all Rpf's or rabbit polyclonal antisera raised against peptides of Rv0867c. This demonstrated the enhanced production of Rv0867c, Rv1884c, and Rv2389c above the background of the BCG homologs of these proteins (FIG. 3A). This supernatant was similarly immunoblotted with rabbit polyclonal antisera to Ag85complex and the overexpression of Rv1886c and Rv3804c was observed (FIG. 3B). The cell pellet was immunoblotted with rabbit polyclonal antisera to Rv3407 and the expression of Rv3407, the homolog of which is normally silent in BCG, was confirmed (FIG. 3C). Cell pellets were also immunoblotted with DosR-specific rabbit polyclonal antisera to demonstrate the overexpression of DosR itself and with rabbit antisera to Rv1733c to demonstrate the upregulation of a DosR regulated protein (FIG. 3D).

In order to complete the construction of this vaccine to make it suitable for human use, the marker cassette of the integrated plasmid was then removed. Electrocompetent AFV-102pRC108 cells were electroporated with plasmid pYUB870hyg, which encodes the γΔ resolvase of Tn1000, a sacB allele, and a hygromycin resistance gene (GenBank #ABD64366). Transformants resistant to both kanamycin and hygromycin were selected on 7H10 media and inoculated into 7H9 liquid media with 10% (v/v) OADC and 0.05% (v/v) tyloxapol and no antibiotics. After seven days growth, dilutions of these liquid cultures were plated on 7H10 containing 10% sucrose to select for recombinants from which the aphA-sacB marker has been excised and the pYUB870hyg plasmid has been lost by dilution and selection against the sacB allele.

Sucrose-resistant transformants were picked for PCR analysis of the integrated antigen cassettes and were inoculated into 7H9 liquid media for immunoblot analysis as before. PCR analysis revealed that the antigen expression cassettes were still present in the chromosome and that the hygromycin resistance marker and sacB gene had been excised. Immunoblotting of supernatants and cell pellets with antisera to Rpf, Ag85 complex, Rv3407, and DosR confirmed that excision of the marker cassette from the chromosome did not effect the expression of the inserted antigen cassettes.

Strain AERAS-407 was constructed by removal of the anti-biotic resistance marker from AFV-102pRC108.

Example 3 Demonstration of the Immunogenicity and Protection Elicited by Vaccination with AERAS-407 in Mice

To illustrate the immune responses elicited by AERAS-407 and to demonstrate its protective efficacy against tuberculosis, four groups of 10 C57/BL6 mice are vaccinated subcutaneously with either 1) saline, 2) BCG, 3) BCG-PfoA or 4) AERAS-407. Mice in groups 2, 3, and 4 receive 5×10⁵ cfu of BCG or recombinant BCG. After 10 weeks, five mice from each group are sacrificed for immune assays and the remaining animals are challenged with 100 cfu aerosolized M. tuberculosis Erdman.

To measure humoral immune responses, a peptide microarray chip has been designed. The chip is spotted with overlapping 50 amino acid peptides generated from the sequences of Rv1886c, Rv3804c, Rv3407c, Rv0867c, Rv1884c, Rv2389c, Rv3133c, and all BCG encoded DosR-regulated proteins which are at least 2 fold induced by the overexpression of DosR (i.e. DosR regulated genes for which transcription is at least 2 fold greater in BCG constitutively expressing the DosR regulon than the wild-type parent strains under aerobic conditions). Pooled sera from each group is incubated with 3 peptide chips for 1 hr at 37° C. The chips are then washed with phosphate-buffered saline (PBS) pH 7.2 and incubated with fluorescein isothiocyanate (FITC) conjugated goat anti-mouse IgG sera (Abcam, Cambridge, Mass.) for 1 hour at 37° C. The chips are then washed again with PBS and the immunofluorescence is read with a Genepix 4000B Array Scanner (Molecular Devices, Sunnyvale, Calif.). Values for each peptide spot are averaged for the 3 chips in each group. Mice receiving AERAS-407 show greater antibody responses to peptides derived from Rv1886c, Rv3804c, Rv3407c, Rv0867c, Rv1884c, Rv2389c and DosR regulated proteins than unvaccinated mice or mice vaccinated with BCG or BCG-PfoA.

To measure cellular immune responses elicited by AERAS-407, spleens are harvested from each and homogenized. Splenocyte concentrations are adjusted to 5×10⁵ cells/well in R10 media in multichamber plates and are incubated for 3 days at 37° C. with purified Rv1886c, Rv3804c, Rv3407c, Rv0867c, Rv1884c, or Rv2389c at a concentration of 1 μg/ml. In addition, splenocytes from each mouse are incubated with Rv2623 and Rv3130c as these proteins are known to be highly upregulated in DosR overexpressing strains and are known to be potent T cell immunogens. After the 3 day incubation, supernatants are harvested from the splenocyte cultures and assayed by ELISA for interferon-γ produced in response to antigen stimulation. Control cultures of unstimulated splenocytes or PMA/PHA stimulated splenocytes are included as negative and positive controls, respectively.

To quantify protection afforded by vaccination with AERAS-407, the 5 remaining mice from each group are sacrificed 10 weeks after challenge with M. tuberculosis and lungs and spleens are aseptically harvested from each animal. Lungs and spleens are homogenized in PBS and dilutions are plated on 7H10 agar. After 4 weeks of incubation, the number of M. tuberculosis colonies are enumerated for each animals lungs and spleen and corrected for the dilution factor. This value is the number of live tubercle bacilli present in the lungs and spleen of each animal. BCG vaccination typically results in approximately a 1 log reduction in cfu/lung and spleen versus saline vaccination. AERAS-407 vaccination results in a greater reduction of Mtb load in the lungs and spleens of mice.

Example 4 Construction of a Second Recombinant BCG Vaccine Genetically Engineered to Over Express Selected Classical M. tb Antigens and the DosR Regulon

A second combination of antigens (TB10.4, Ag85B, Ag85A and Rv3407) was also overexpressed in the rBCG AFV102 strain by integration into the chromosome at the attB site. This was carried out in a manner similar to that described in Example 2 for the Aeras 407 construct. To integrate this second antigen set, the integration plasmid pAF707 (depicted schematically in FIG. 4) was constructed as described for pJFINT in Example 2 except for the following differences: a hygromycin resistance gene was used in place of the kanamycin resistance gene, and the PBlaf promoter was used to drive antigens TB10.4, Ag85B, Ag85A and Rv3407 for over expression in the order listed. All other general materials and methods were similar to those described in Example 2. The resultant strain was designated Aeras 406. The final strain was further analyzed by PCR followed by sequencing analysis and confirmed to have the desired genotype. The strain was further demonstrated to overexpress the selected antigens by SDS-PAGE analysis and immunoblot testing as described in Example 2 (data not shown).

Example 5 Protection of Non-human Primates from Tuberculosis by Vaccination with AERAS-407

The protective efficacy of AERAS-407, particularly in relation to the enhanced capacity to prevent latent infection and reactivation of disease, is best tested in rhesus macaques as mice do not create granulomatous latent foci as humans and other primates do. To evaluate the protective efficacy of AERAS407 in NHP's, four groups of six weight and sex-matched rhesus macaques are vaccinated with 1) saline, 2) BCG 1331, 3) BCG-Pfo, or 4) AERAS-407. Each animal in groups 2-4 receives 5×10⁵ cfu of the respective BCG or rBCG by intradermal injection. Fifteen weeks after vaccination, all animals are challenged by bronchial installation of approximately 300 cfu of M. tuberculosis Erdman. All animals are evaluated monthly for six months for clinical symptoms of tuberculosis by chest X-ray, weight, feeding, cough, lethargy, and immune responses to TB specific proteins. All animals that die during the six month observation period are necropsied and tissue pathology and Mtb burden by organ is measured as in Example 3. All moribund animals are humanely euthanized and similarly examined. Six months post-challenge all surviving animals are euthanized and necropsied for tissue pathology and Mtb burden in lungs, liver and spleen. AERAS-407 vaccination results in decreased mortality, decreased tissue damage and lower counts of viable Mtb organisms in the lungs of experimentally infected animals.

While the invention has been described in terms of its preferred embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments as described above, but should further include all modifications and equivalents thereof within the spirit and scope of the description provided herein. 

1. A recombinant Bacille Calmette-Guerin (BCG) wherein at least one DosR regulon gene is up-regulated; and said recombinant BCG comprises at least two nucleic acid sequences which are different from each other, each of which encodes one or more genes that are overexpressed, said at least two nucleic acid sequences including i) a first nucleic acid sequence encoding at least one Mycobacterium tuberculosis (Mtb) antigen; and ii) a second nucleic acid sequence encoding at least one Mtb reactivation antigen, wherein said second nucleic acid sequence is not a dosR antigen.
 2. The recombinant BCG of claim 1, wherein one or more of said at least two nucleic acid sequences is integrated into a chromosome of said rBCG.
 3. The recombinant BCG of claim 1, wherein one or more of said at least two nucleic acid sequences is an extrachromosomal nucleic acid sequence.
 4. The recombinant BCG of claim 1, wherein said at least one Mtb antigen is 85A.
 5. The recombinant BCG of claim 1, wherein said at least one Mtb antigen is 85B.
 6. The recombinant BCG of claim 1, wherein said at least one Mtb reactivation antigen is selected from the group consisting of Rv0867c, Rv1009, Rv1884c, Rv2389c, Rv2450c, Rv0288, Rv0685, Rv0824c, Rv2744c, Rv3347c, Rv1130 and Rv1169c.
 7. The recombinant BCG of claim 1, wherein said at least one Mycobacterium tuberculosis (Mtb) antigen is selected from the group consisting of Rv2623, Rv2626c, Rv2005c, Rv1996, Rv2029c, Rv2627c, Rv1908, Rv3837, Rv2780 and Rv1349.
 8. The recombinant BCG of claim 1 , wherein expression of said at least one DosR regulon gene is up-regulated by enhanced promoter activity.
 9. The recombinant Bacille Calmette-Guerin (BCG) of claim 1, wherein said at least one DosR regulon gene that is up-regulated is selected from the group consisting of Rv1738, Rv2623, Rv2031c, Rv2032, Rv2626c, Rv2005c, Rv3127, Rv1733c, Rv1996, Rv2628, Rv0079, Rv3130c, Rv3131, Rv1813c, Rv2006, Rv2029c, Rv2627c, Rv2030c, Rv3132c, and Rv2629.
 10. The recombinant BCG of claim 6, wherein said at least one Mtb reactivation antigen is selected from the group consisting of Rv0867c, Rv1009, Rv1884c, Rv2389c and Rv2450c.
 11. The recombinant BCG of claim 10, wherein said at least one Mtb reactivation antigen is selected from the group consisting of Rv1884c, Rv2389c, and Rv2450c.
 12. A recombinant Bacille Calmette-Guerin (BCG) comprising at least a first nucleic acid sequence and a second nucleic acid sequence which are different from each other, each of which encodes one or more genes that are overexpressed, wherein said first nucleic acid sequence encodes an antigen selected from the group consisting of Rv2450c, Rv1009, Rv0867c, Rv2389c, Rv1884c, Rv0288, Rv0685, Rv0824c, Rv2744, Rv3347c, Rv1130, Rv1169c; and wherein said second nucleic acid sequence encodes an antigen selected from the group consisting of Rv1738, Rv2623, Rv2031c, Rv2032, Rv2626c, Rv2005c, Rv3127, Rv1733c, Rv1996, Rv2628, Rv0079, Rv3130c, Rv3131, Rv1813c, Rv2006, Rv2029c, Rv2627c, Rv2030c, Rv2629, Rv2450c, Rv1009, Rv0867c, Rv2389c, Rv1884c, Rv0288, Rv0685, Rv0824c, Rv2744, Rv3347c, Rv1130, Rv1169c, Rv1886, Rv1980c, Rv3804c, Rv3875, Rv1926c, Rv0467, Rv3873, Rv1908c, Rv1174c, Rv2780, Rv2620c, Rv1793, Rv1349 and Rv3132; and wherein at least one DosR regulon gene is up-regulated in said recombinant BCG.
 13. The recombinant BCG of claim 12, wherein said at least one Mtb antigen is a reactivation antigen selected from the group consisting of Rv0867c, Rv1009, Rv1884c, Rv2389c and Rv2450c.
 14. The recombinant BCG of claim 12, wherein said at least one Mtb antigen is a reactivation antigen selected from the group consisting of Rv1884c, Rv2389c, and Rv2450c.
 15. The recombinant Bacille Calmette-Guerin (BCG) of claim 12, wherein said at least one DosR regulon gene that is up-regulated is selected from the group consisting of Rv1738, Rv2623, Rv2031c, Rv2032, Rv2626c, Rv2005c, Rv3127, Rv1733c, Rv1996, Rv2628, Rv0079, Rv3130c, Rv3131, Rv1813c, Rv2006, Rv2029c, Rv2627c, Rv2030c, Rv3132c, and Rv2629. 